Bacterial endotoxins are produced by Gram negative bacteria, many of which are very dangerous or deadly in human beings and animals. In periodontal pockets, Gram-negative bacteria, their endotoxins and other inflammatory agents cause gingivitis and other periodontal diseases It is important to identify the existence of endotoxins and, if possible, the concentration of the endotoxins in the periodontal pockets in order to promptly initiate the proper medical and dental treatment. The monitoring of endotoxins in the periodontal pockets of a subject may also be used to diagnose periodontal diseases and the effect of treatment programs.
It has been known for many years that bacterial endotoxins activate the blood clotting enzyme occurring in amebocyte lysates of horseshoe crabs. This property has been described in many publications, e.g. J. Levin and F. B. Bangs, 1968, Thomb. Diath. Haemonth, 19:186 and S. Nakamura, 1976, Biochemical and Biophysical Research Communication, 72:902. These investigations demonstrated that the coagulation of Limulus amebocyte lysate (LAL) requires activation of the proclotting enzyme by endotoxin in the presence of divalent cations. The active enzyme thus formed cleaves coagulogen at the C-carboxyl terminal between the glycine and arginine subunits. The cleaved coaculogen molecules polymerize and thus bring about coagulation. The first endotoxin tests were based on observation of the gel thus formed. The gelation reaction test method is deficient for use in monitoring periodontal disease because of the considerable length of time necessary to carry it out, because it does not accurately measure endotoxin concentration, because it is difficult to standardize and because it requires highly skilled experienced personnel to perform it.
Fluorogenic or chromogenic substrates have been used for the assay of many enzymes, e.g. trypsin, thrombin, thromboplastin, serine protease, plasmin and plasminogen. It was also realized that fluorogens or chromogens can be used as LAL-substrates. See e.g. Iwanaga, et al., 1978; Homeostatis, 7:183 and patent publications U.S. Pat. No. 4,188,265, U.S. Pat. No. 4,406,832 and U.S. Pat. No. 4,510,241. The use of chromogenic substrates nas become a means to both study and clinically monitor various enzymes and inhibitors in the complex coagulation processes of man. An extensive list of enzyme specific substrates are commercially available for measuring enzymes such as trypsin, thrombin, thromboplastin, plasmin, plasmin kallikrein, urokinase, and plasminogen. Iwanaga, et al., Hemostasis 7:183-188 (1978) discloses that synthetic substrates can be used to measure the level of endotoxin activated pro-clot enzyme in LAL prepared from the blood of both the Japanese (Tachypleus tridentatus) and the American (Limulus polyphemus) horseshoe crabs.
Chromogenic substrate methods for assaying bacterial endotoxins using Limulus amebocyte lysate are also described in pages 209-220 of "Biomedical Applications of the Horseshoe Crab" (1979) Allen R. Liss, Inc. and U.S. Pat. No. 4,301,245. However, these disclosures are directed to the testing of blood which may contain endotoxins or the testing of food for endotoxin contamination.
There are three methods commonly used for monitoring bacteria in a periodontal pocket:
1. Dark field or phase microscopy; PA1 2. Cultural microbiological evaluation; and PA1 3. Fluorescent antibody identification. PA1 a) obtaining a sample from the periodontal pocket of the subject; PA1 b) contacting the sample with an amebocyte lysate under conditions so as to activate an enzyme capable of cleaving a bond between an arginyl group and a nitrogen-containing group; PA1 c) contacting the activated enzyme with a substrate comprising an arginyl group and a suitable nitrogen-containing group bound to the arginyl group so as to form an amine; PA1 d) treating the resulting amine to produce a detectable product; and PA1 e) quantitatively determining the amount of product formed and thereby the amount of bacterial endotoxins present in the periodontal pocket.
The first method is inaccurate, time consuming, and requires the knowledge of a specialist. The second method is expensive and too cumbersome to be undertaken by the dentist in his office. The third method is also difficult for the dentist, and too expensive to be performed at each individual periodontal pocket site.
With the present method, it is possible to conduct the desired endotoxin test within a short period of time in a dental office and without the need for a specialist or expensive equipment. The time period for the method of the present invention is much shorter than that required for previous test methods for bacterial endotoxins. Moreover, the present method simply and accurately measures the concentration of the endotoxin to indicate the extent of the Gram negative bacterial infection in the periodontal pocket.